The present invention relates to a novel method for non-radioactive detection of membrane-bonded nucleic acids, including nucleic acids that, for instance, contain single nucleotide polymorphisms (SNPs), DNA arrays (cosmids, yeast artificial chromosomes (YAC""s), bacterial artificial chromosomes (BAC""s), cDNA""s, PCR fragments, oligonucleotides), RNA arrays and all nucleic acid fragments that are transferred from gels (agarose or polyacrylamide (PAA) gels) to membranes, including genomic DNA/plasmid DNA fragments (Southern) and mRNAs (Northern) and a test kit to apply the method.
Arrays are individual nucleic acids preferably arranged to a defined degree on a membrane in rasters.
The identification of exchanges of individual bases within DNA (SNP""s) is of great importance to investigating the causes of complex diseases, e. g. hypertension, and for diagnostic purposes (Lander, E. S. (1996): Science 274, 536-539, Collins, F. S. et al. (1997) Science 278, 1580-1581, Marshall E. (1997): Science 277, 1752-1753). Such an exchange is may be detected by sequencing the interesting regions.
Traditionally the sequence is determined by the enzymatic chain-terminating method where the sequencing products are marked by the incorporation of radioactively or non-radioactively labeled nucleotides. Following gel electrophoretic separation the sequence may be determined by evaluation of the sequence gels. Direct labelling of the sequencing products, however, has the disadvantage that only one single DNA fragment may be analyzed per sequencing batch.
Multiplex technology (see EP-A 0 303 459) overcomes this disadvantage by simultaneously sequencing a few DNA molecules. The resulting mixtures of sequencing products formed are gel electrophoretically separated, transferred to a nylon membrane and fixed there. By hybridizing this membrane with a probe which is specific for a single DNA fragment the sequences of a few DNA fragments may be successively read using the same membrane. However, a big disadvantage of this method is that the detection of various DNA fragments is effected by using radioactively labelled probes. That is why automation of the whole process to further increase the flow rate will not be feasible.
Furthermore, a non-radioactive detection method is known which is described by Richterich and Church (Richterich, R., Church, G. M (1993) Methods Enzymol 218: 187-222), however showing the disadvantages to be not suitable for successive hybridizations and usually hybridizations may not be carried out exclusively at room temperature.
The objective of the present invention is to establish an easy to perform non-radioactive method to be applied which maintains or even improves, on the one hand, the sensitivity of radioactive methods and, on the other hand, allows an immediate, direct successive detection.
The invention is implemented according to the claims. Surprisingly, this task may be solved by means of a method which may be applied in all stages at room temperature. The method according to the invention is suited for a non-radioactive detection of membrane-bound nucleic acids (A) by hybridizing NAs bound to a membrane which are obtained by means of methods known per se with a special hybridization buffer containing Tris HCl, Tris base, NaCl, Triton X-100, SDS and a blocking reagent and subsequently detecting the bound NA. The invention does not include the detection of sequencing products according to the multiplex method in accordance with EP 303 459 (G. Church).
Preferably membrane-bound DNA is detected.
The method is preferably characterized by the following steps:
1. Prehybridization of a membrane containing a DNA according to a method known per se, preferably in a dish, with the aid of a special hybridization buffer,
2. hybridization of the membrane by means of a 5xe2x80x2biotinylated probe in the same buffer,
3. repeated washing of the membrane with buffer I containing PBS (Na2HPO4, NaH2PO4, NaCl, pH approx. 7.3), SDS and blocking reagent,
4. incubation of the membrane with streptavidin alkaline phosphatase conjugate in buffer I,
5. one-time washing of the membrane with buffer I, thereupon repeated washing with buffer II, containing PBS and SDS and repeated equilibration at pH 9.75 with buffer III containing Tris-HCI and diethanol amine,
6. after equilibration, transfer of the membrane into a substrate solution consisting of CDPstar(copyright) (disodium 2-chloro-5-(4-methoxyspiro{1,2-dioxetane-3,2xe2x80x2-(5-chloro)-tricyclo[3.3.1.13,7]decan}-4-yl)-1-phenyl phosphate, Tropix(copyright) and buffer III and, if necessary, agitation of the membrane to reach a uniform distribution of the substrate solution,
7. after short drainage, fixing of the membrane, e.g. onto a piece of plastic, and covering, preferably with a transparent foil,
8. exposing, preferably by means of an X-ray film or a CCD camera,
9. after exposure returning of the membrane and repeated stripping with a preheated buffer IV containing EDTA and SDS,
10. one-time washing with buffer V containing Tris-HCl and NaCl.
The evaluation is performed according to established methods such as e.g. the xe2x80x98skilled pattern analysisxe2x80x99 principle.
The most important step in the above method is hybridization with a special hybridization buffer, thereby allowing repeated subsequent hybridization and a sensitivity equivalent to or better than radioactive detection is obtained. Thus, the method according to the invention for the non-radioactive detection of nucleic acids containing e.g. xe2x80x98single nucleotide polymorphismsxe2x80x99 (SNP""s) is to be regarded as a great improvement. In addition, the use of radioactivity which is hazardous to health may be avoided. Apart from that, by summarizing otherwise time-consuming intermediate steps and optimizing the compositions of solutions and the necessary washing parameters constant results may be achieved allowing to provide the basis for the future development of automated detection.
The test kit according to the present invention for the non-radioactive detection of nucleic acids containing e.g. xe2x80x98single nucleotide polymorphismsxe2x80x99 (SNPs) contains the following solutions:
Hybridization buffer: 5 l
500 ml 10xc3x97TNT
1250 ml of 20% SDS
10 g of blocking reagent (Boheringer Mannheim, DIG kit)
3250 ml of H2O (twice distilled)
10xc3x97PBS: 5 l
516.2 g of Na2HPO4 
132.6 g of NaH2PO4 
198.7 g of NaCl
H2O to 5 l pH: approx. 7.3
Buffer I: 5 l
10 g of blocking reagent
250 ml 10xc3x97PBS
125 ml of 20% SDS
4625 ml of H2O (twice distilled), to be heated for 8 min. in a microwave oven, to be stirred until the blocking reagent is dissolved.
Buffer II: 5 l
250 ml 10xc3x97PBS
125 ml of 20% SDS
4625 ml of H2O (twice distilled)
10xc3x97buffer III: 2 l
64.4 g of Tris HCl
1770 ml of H2O (twice distilled)
210.4 g of diethanol amine pH: 9.75
Buffer IV: 5 l
4700 ml of H2O (twice distilled)
50 ml of 0.5 M EDTA, pH 8.0
250 ml of 20% SDS
Buffer V: I 1 l
920 ml of H2O (twice distilled)
30 ml of 5 M NaCl
50 ml of 1 M TrisCl, pH 8.0
10xc3x97TNT: 1 l 44.4 g of Tris HCl
25.6 g of Tris base
73 g of NaCl
818 ml of H2O ( )
100 ml of Triton X-100 pH: approx. 8.0